Stimulation of G_q-coupled M1 muscarinic receptor causes reversible spectrin redistribution mediated by PLC, PKC and ROCK

JCS02872 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Muscarinic agonist-induced spectrin redistribution depends on transient activation of ROCK. (A) Proteolytic conversion from the full-length (*) to C-terminally cleaved, active ROCK (**) is not significantly induced in M1-CHO cells stimulated with 5 mM Oxo-M for 5 minutes. The results of two separate experiments (Exp.1 and 2) are shown. The relative amount of ROCK fragment (cleaved/full-length ROCK) was 0.25, 0.17 and 0.70 for  0, 5 and 60 minutes, respectively in Exp 1.

• Movie 1 -

  Movie 1. Muscarinic agonist-induced reversible redistribution of YFP-aII spectrin accompanied with membrane blebbing. A M1-CHO cell expressing YFP-aII spectrin (green) was stimulated with 10 mM Oxo-M. Notice that YFP-aII spectrin redistributed along with bleb formation and re-established its pre-stimulus position upon withdrawal of blebs. Images were captured every 5 seconds over a 15-minute period, and converted into a 753 real-time movie. A period of Oxo-M application is indicated in the movie. The movie corresponds to the data presented in Fig. 3. Bar, 10 mm.

• Movie 2 -

  Movie 2. Ionomycin-induced irreversible redistribution of YFP-aII spectrin. A M1-CHO cell expressing YFP-aII spectrin (green) was stimulated with 5 mM ionomycin. Notice that YFP-aII spectrin redistributed fuses into the cytosol after blebbing. Images were captured every 5 seconds over a 14-minute period, and converted into a 753 real-time movie. A period of ionomycin application is indicated in the movie. The movie corresponds to the data presented in Fig. 5. Bar, 10 mm.

• Movie 3 -

  Movie 3. Reversible and global aII spectrin redistribution caused by changes in osmotic pressure. M1-CHO cells expressing YFP-aII spectrin (green) were incubated in the isotonic or hypotonic solution in the following sequence: isotonic (5 minutes), hypotonic (10 minutes) and isotonic (10 minutes). Notice that spectrins were evenly redistributed without blebbing morphology. Frames were captured every 5 seconds over a 25-minute period, and converted into a 753 real-time movie. A period of incubation in the hypotonic solution is indicated in the movie. Bar, 10 mm.

• Movie 4 -

  Movie 4. Altered distribution of aII spectrin by cytochalasin D and impaired responsiveness to Oxo-M stimulation. M1-CHO cells expressing YFP-aII spectrin (green) were first stimulated with 10 mM Oxo-M to check responsiveness of the cells. Following 15 minutes incubation with cytochalasin D, cells were similarly stimulated with Oxo-M. Notice that cytochalasin D treatment completely disrupted the distribution of aII spectrin at the cell periphery resulting in concentrated and static foci. Images were captured every 5 seconds over a 20-minute period (10 minutes for each stimulation sequence), and converted into a 753 real-time movie. A break in the movie presents a 15-minute incubation of cytochalasin D between the first and second Oxo-M application. Duration of Oxo-M application is indicated in the movie. Bar, 20 mm.